1) Structural Basis for fatty acid recognition and transfer by eukaryotic integral membrane plamitoyltransferases - Close to a thousand cellular proteins are modified by posttranslational S-acylation of cysteines, commonly known as protein palmitoylation. Unlike other lipid attachments, which are thought to be permanent, palmitoylation can be reversed by cellular thioesterases, enabling dynamic modulation of the local hydrophobicity of substrate proteins. In humans, palmitoylation is catalyzed by 23 members of the DHHC family of integral membrane enzymes, which contain a signature Asp-His-His-Cys (DHHC) motif. DHHC enzymes use fatty acyl coenzyme A (predominantly the 16 carbon palmitoyl-CoA) to generate an acyl-enzyme intermediate from which the acyl chain is subsequently transferred to a substrate. With a recent systems-level analysis suggesting that more than 10% of the proteome is palmitoylated, the complexity of protein palmitoylation approaches that of protein phosphorylation and ubiquitylation. Yet, fundamental aspects of DHHC enzymes, including their mechanism of catalysis and acyl-CoA binding and recognition, have been challenging to analyze without detailed structural information. To obtain insights into the structural mechanism of DHHC enzymes, we solved the crystal structures of two DHHC family members: human DHHC20 and a catalytically inactive mutant of zebrafish DHHC15. We also solved the structure of human DHHC20 conjugated to an irreversible inhibitor that mimics the acylated enzyme intermediate. These structures and the subsequent biochemical experiments lead to a model where the four transmembrane helices of hDHHC20 and zfDHHS15 form a tepee-like structure with the active site, contained in the highly conserved cytosolic DHHC cysteine-rich domain, at the membrane-cytosol interface. Our structures readily explain why candidate cysteines for palmitoylation are proximal to the membrane. The transmembrane domain forms a cavity where the acyl chain of acyl-CoA is inserted. Cavity-lining residues are determinants of fatty acyl recognition and chain-length selectivity. Our structures enabled us to engineer mutants of human DHHC20 with altered acyl chain length selectivities. The structural results form an important seed for the development of structure-based tools such as orthogonal DHHC enzyme-fatty acyl CoA pairs that will help investigate the enzyme-substrate network of this biologically and biomedically important family of enzymes. These results will also enable discovery of small molecule probes that will be important tools for understanding the biology of DHHC enzymes as well as plausible starting point for developing novel therapeutic strategies. Given that human DHHC20 palmitoylates EGFR and has been proposed as a target for cancer therapy, our work has important biomedical relevance. 2) In Vitro reconstitution and biochemical studies of iron transport into mitochondria - In this project, we are focusing on transporters that move iron across organellar membranes. One transporter of interest is Mitoferrin, the only known major transporter of iron into mitochondria. Subsequently, the iron is utilized in the biosynthesis of heme, a central component in hemoglobin, myoglobin, and cytochromes , and in the biosynthesis of iron-sulfur clusters, important cofactors involved in a wide range of cellular activities, viz. electron transport in respiratory chain complexes, regulatory sensing, photosynthesis and DNA repair. Mitochondria thus play a central role in the cellular utilization and balance of iron. The malfunction of this balance can lead to several diseases such as sideroblastic anemia. Mitoferrin was proposed as an iron transporter from genetic and cell-based studies but the iron transport activity have never been demonstrated through an in vitro assay. It belongs to the mitochondrial carrier (MC) family and is atypical given its putative metallic cargo; most MCs transport nucleotides, amino acids, or other small- to medium-size metabolites. To bridge this knowledge gap, we have purified recombinant Mitoferrin-1 and probed its metal ion-binding and transport functions. In order to do so, we had to set up a robust in vitro iron transport assay since there were no reliable in vitro reconstituted transport assays for iron transporters, despite the pivotal importance of iron in biology. This assay has enabled us to investigate the metal ion promiscuity of Mitoferrin and the importance of highly conserved residues in its function. Our studies demonstrated, for the first time that Mitoferrin-1 can transport iron. Mfrn1 can also transport manganese, cobalt, copper, and zinc but discriminates against nickel. Experiments with candidate ligands for cellular labile iron revealed that Mitoferrin-1 transports free iron and not a chelated iron complex and selects against alkali divalent ions. Isothermal titration calorimetry studies showed that Mitoferrin-1 has micromolar affinity for Fe(II), Mn(II), Co(II), and Ni(II). Extensive mutagenesis identified multiple residues that are crucial for metal binding, transport activity, or both. There is a clear abundance of residues with side chains that can coordinate first-row transition metal ions, suggesting that these could form primary or auxiliary metal-binding sites during the transport process. Currently we are pursuing high-resolution structural studies of Mitoferrin that will lead to an atomic level understanding of its mechanism.